Debottlenecking the DOPA 4,5-dioxygenase step with enhanced tyrosine supply boosts betalain production in Nicotiana benthamiana.

Synthetic biology provides emerging tools to produce valuable compounds in plant hosts as sustainable chemical production platforms. However, little is known about how supply and utilization of precursors is coordinated at the interface of plant primary and specialized metabolism, limiting our ability to efficiently produce high levels of target specialized metabolites in plants. L-Tyrosine is an aromatic amino acid precursor of diverse plant natural products including betalain pigments, which are used as the major natural food red colorants and more recently a visual marker for plant transformation. Here, we studied the impact of enhanced L-tyrosine supply on the production of betalain pigments by expressing arogenate dehydrogenase (TyrA) from table beet (Beta vulgaris, BvTyrAα), which has relaxed feedback inhibition by L-tyrosine. Unexpectedly, betalain levels were reduced when BvTyrAα was co-expressed with the betalain pathway genes in Nicotiana benthamiana leaves; L-tyrosine and 3,4-dihydroxy-L-phenylalanine (L-DOPA) levels were drastically elevated but not efficiently converted to betalains. An additional expression of L-DOPA 4,5-dioxygenase (DODA), but not CYP76AD1 or cyclo-DOPA 5-O-glucosyltransferase, together with BvTyrAα and the betalain pathway, drastically enhanced betalain production, indicating that DODA is a major rate-limiting step of betalain biosynthesis in this system. Learning from this initial test and further debottlenecking the DODA step maximized betalain yield to an equivalent or higher level than that in table beet. Our data suggest that balancing between enhanced supply ("push") and effective utilization ("pull") of precursor by alleviating a bottleneck step is critical in successful plant synthetic biology to produce high levels of target compounds.

VAS1-mediated nitrogen reshuffling in aromatic amino acid homeostasis.

Aromatic amino acids (AAAs) are essential for synthesis of proteins and numerous plant natural products, yet how plants maintain AAA homeostasis remains poorly understood. Wu et al. reported that the aminotransferase VAS1 plays a role in AAA homeostasis by transferring nitrogen from AAAs to non-proteinogenic amino acids, 3-carboxytyrosine and 3-carboxyphenylalanine.

Aspergillus oryzae PrtR alters transcription of individual peptidase genes in response to the growth environment.

Aspergillus oryzae PrtR is an ortholog of the transcription factor PrtT, which positively regulates the transcription of extracellular peptidase genes in Aspergillus niger and Aspergillus fumigatus. To identify the genes under the control of PrtR and elucidate its regulatory mechanism in A. oryzae, prtR gene disruption mutants were generated. The control strain clearly showed a halo on media containing skim milk as the nitrogen source, whereas the ΔprtR strain formed a smaller halo. Measurement of acid peptidase activity revealed that approximately 84% of acidic endopeptidase and 86% of carboxypeptidase activities are positively regulated by PrtR. As the transcription of the prtR gene varied depending on culture conditions, especially with or without a protein substrate, it was considered that its transcription would be regulated in response to a nitrogen source. In addition, contrary to previous expectations, PrtR was found to act both in promoting and repressing the transcription of extracellular peptidase genes. The mode of regulation varied from gene to gene. Some genes were regulated in the same manner in both liquid and solid cultures, whereas others were regulated in different ways depending on the culture conditions. Furthermore, PrtR has been suggested to regulate the transcription of peptidase genes that are closely associated with other transcription factors. KEY POINTS: • Almost all peptidase genes in Aspergillus oryzae are positively regulated by PrtR • However, several genes are regulated negatively by PrtR • PrtR optimizes transcription of peptidase genes in response to culture conditions.

One-Leg Robotic-Assisted Gait Training Efficiently Improves Gait Independence for Acute Stroke Hemiplegic Patients: A Prospective Pilot Study.

ABSTRACT: Welwalk is a one-leg robotic-assisted gait system for stroke hemiplegic patients. This study examined the feasibility and efficacy of gait training using Welwalk (Welwalk training) for hemiplegic patients in the early phase after stroke onset, via cooperation between acute care and rehabilitation hospitals. Seven acute stroke patients (mean number of days from onset = 7.9) with severe lower extremity paralysis participated. Patients underwent Welwalk training for 40 min/d, 5 d/wk in an acute care hospital, then 7 d/wk in a rehabilitation hospital with a seamless transition. Functional Independence Measure scores for walking were assessed weekly. The endpoint was reaching Functional Independence Measure walk score of 5 (supervision level). The primary outcome was improvement efficiency of Functional Independence Measure walk, which was the increase in Functional Independence Measure walk score divided by the number of weeks required. Functional Independence Measure walk score for all patients improved from 1.1 to 5 ( P = 0.01, r = 0.96). The mean number of weeks to achieve Functional Independence Measure walk score of 5 was 5 wks, and the improvement efficiency of Functional Independence Measure walk had a mean value of 0.9. No adverse events were reported during Welwalk training. Hemiparetic patients' gait independence may be safely and rapidly improved by starting Welwalk training in the early phase after stroke onset.

Population Pharmacokinetics of the Novel Adenosine A2A Antagonist/Inverse Agonist KW-6356 and Its Active Metabolite Following Single and Multiple Oral Administration in Healthy Individuals and Patients with Parkinson's Disease.

KW-6356 is a selective antagonist and inverse agonist of the adenosine A2A receptor. The primary aim of the present analysis was to characterize the pharmacokinetics (PK) of KW-6356 and its active metabolite M6 in healthy subjects and patients with Parkinson's disease (PD). We pooled concentration-time data from healthy subjects and patients with PD who were administered KW-6356. Using these data, we developed a population PK model by sequentially fitting the KW-6356 parameters followed by the M6 parameters. A first-order absorption with a 1-compartment model for KW-6356 and a 1-compartment model for M6 best described the profiles. The covariates included in the final models were food status (fed/fasted/unknown) on first-order absorption rate constant, baseline serum albumin level on apparent clearance of KW-6356, and baseline body weight on apparent volume of distribution of KW-6356 and apparent clearance of M6. No covariate had a clinically meaningful impact on KW-6356 or M6 exposure.

Wearable Integrated Volitional Control Electrical Stimulation Device as Treatment for Paresis of the Upper Extremity in Early Subacute Stroke Patients: A Randomized Controlled Non-inferiority Trial.

OBJECTIVE: To investigate the effect of a wearable integrated volitional control electrical stimulation (WIVES) device that has been developed as more compact and simpler to use in daily life compared with conventional integrated volitional control electrical stimulation (IVES) devices. DESIGN: Randomized controlled non-inferiority trial. SETTING: Convalescent rehabilitation ward. PARTICIPANTS: Patients with paresis of the upper extremity (UE) after early subacute stroke (N=20). INTERVENTIONS: Eligible patients were randomized to receive IVES treatment or WIVES treatment for 8 hours per day for 28 days in daily living, in addition to standard rehabilitation treatment. In both groups, the extensor digitorum communis on the affected side was the target muscle for stimulation. MAIN OUTCOME MEASURE: Primary outcomes were assessed with Fugl-Meyer Assessment of the UE (FMA-UE) before and after treatment. Non-inferiority was determined with a specified margin of non-inferiority. RESULTS: Twenty patients completed the trial (IVES group: n=10, WIVES group: n=10). FMA-UE improved in both groups. The mean change in FMA-UE was 4.7 for the IVES group and 6.0 for the WIVES group (P>.05, 95% confidence interval: -6.73 to 4.13). The mean difference between the groups was 1.3, and the upper 95% confidence interval did not exceed the non-inferiority margin. CONCLUSION: The effectiveness of WIVES treatment is non-inferior to that of IVES treatment. As a portable device, IVES may facilitate the use of affected upper extremities in daily living and may help improve paresis of the UE.

Coordinated regulation of the entry and exit steps of aromatic amino acid biosynthesis supports the dual lignin pathway in grasses.

Vascular plants direct large amounts of carbon to produce the aromatic amino acid phenylalanine to support the production of lignin and other phenylpropanoids. Uniquely, grasses, which include many major crops, can synthesize lignin and phenylpropanoids from both phenylalanine and tyrosine. However, how grasses regulate aromatic amino acid biosynthesis to feed this dual lignin pathway is unknown. Here we show, by stable-isotope labeling, that grasses produce tyrosine >10-times faster than Arabidopsis without compromising phenylalanine biosynthesis. Detailed in vitro enzyme characterization and combinatorial in planta expression uncovered that coordinated expression of specific enzyme isoforms at the entry and exit steps of the aromatic amino acid pathway enables grasses to maintain high production of both tyrosine and phenylalanine, the precursors of the dual lignin pathway. These findings highlight the complex regulation of plant aromatic amino acid biosynthesis and provide novel genetic tools to engineer the interface of primary and specialized metabolism in plants.

Accurate diagnosis of sarcopenia without using a body composition analyzer in a convalescent rehabilitation ward.

Hishikawa N, Sawada K, Shono S, Sakurai M, Yokozeki M, Maeda H, Ohashi S, Ueshima K, Mikami Y. Accurate diagnosis of sarcopenia without using a body composition analyzer in a convalescent rehabilitation ward. Jpn J Compr Rehabil Sci 2023; 14: 26-32. Objective: The Asian Working Group for Sarcopenia 2019 recommends diagnosing sarcopenia without using a body composition analyzer and initiating treatment early. The present study aimed to investigate the accuracy of diagnosing sarcopenia without a body composition analyzer in a convalescent rehabilitation ward. Methods: Eighty-five patients admitted to a convalescent rehabilitation ward were included, and sarcopenia diagnoses were performed with and without a body composition analyzer. To assess the accuracy of diagnosing sarcopenia without using a body composition analyzer, sensitivity, specificity, positive predictive value, and negative predictive value were calculated relative to sarcopenia diagnoses made using a body composition analyzer. Results: The sensitivity of the technique for diagnosing sarcopenia was 0.94, specificity was 0.77, positive predictive value was 0.86, and negative predictive value was 0.90. Conclusion: The accuracy of diagnosing sarcopenia without using a body composition analyzer was high. However, this technique may miss sarcopenia cases in patients with increased calf circumference due to adipose tissue and/or edema.

Effects of vitamin B12 in culture medium for calcified nodule formation by rat dental pulp cells.

Background/purpose: Tooth or bone regeneration requires large numbers of mesenchymal stem cells (MSCs). Although dental pulp is a suitable cell source, the number of MSCs present in this tissue is limited and they require a long period for regeneration. Therefore, the present study investigated vitamin B12 (Vb12) as an osteoinductive factor for MSCs obtained from dental pulp. Materials and methods: Dental pulp tissue was removed from the root canals of the extracted mandibular incisors of three 6-week-old male Fischer 344/N Slc rats using an endodontic file and whole cells were harvested. After the primary culture, cells were sub-cultured for calcified nodule formation in MEM containing dexamethasone (Dex), ß-glycerophosphate (ß-GP), vitamin C (Vc), and Vb12. Calcified nodules were confirmed under an inverted phase-contrast microscope. The alkaline phosphatase (ALP) activity of cells and quantity of Ca2+ in calcified nodules were measured. Results were analyzed using the Tukey-Kramer test. Results: Densely arranged calcified nodules were microscopically observed after the subculture of cells with Dex, ß-GP, Vc, and Vb12. The ALP activity level was 0.077 ± 0.023 µmol/µg DNA in MEM supplemented with Vb12, which did not significantly differ from that without Vb12. A mass of calcium nodules formed in culture medium containing Dex, ß-GP, Vc, and Vb12. The quantity of Ca2+ increased from 13.04 ± 0.44 to 20.91 ± 0.56 mg/dL (P < 0.01). Conclusion: Vb12 is effective for in vitro tooth or bone regeneration by the MSCs of rats and is useful as an osteoinductive factor for MSCs.

Safety, Tolerability, and Pharmacokinetics of the Novel Adenosine A2A Antagonist/Inverse Agonist KW-6356 Following Single and Multiple Oral Administration in Healthy Volunteers.

KW-6356 is an adenosine A2A receptor-selective antagonist and inverse agonist. We conducted 2 studies: study 6356-001 (no NCT number), a randomized, double-blind, placebo-controlled phase 1 trial of single ascending (1, 3, 10 mg) and multiple (6 mg once daily) oral doses of KW-6356 in healthy Japanese subjects; and study 6356-004 (NCT03830528), including a randomized, double-blind, placebo-controlled phase 1 trial of single ascending (21, 42, 60 mg) and multiple (24 mg once daily) oral doses of KW-6356, and a phase 1 open-label trial of multiple oral doses (6 mg once daily) of KW-6356 in healthy Japanese and White subjects, to evaluate the safety, tolerability, and pharmacokinetics of KW-6356. KW-6356 was well tolerated after administration of single doses of up to 60 mg and multiple doses of up to 24 mg once daily for 14 days. The pharmacokinetics of KW-6356 were linear after a single dose of up to 60 mg KW-6356. The mean terminal elimination half-life of KW-6356 ranged from 18.4 to 43.1 hours following administration of single doses of 1-60 mg. There was no clear difference in the safety or pharmacokinetics of KW-6356 between healthy Japanese and White subjects.

Biochemical characterization of plant aromatic aminotransferases.

Aromatic aminotransferases (Aro ATs) are pyridoxal-5-phosphate (PLP)-dependent enzymes that catalyze the transamination reactions of an aromatic amino acid (AAA) or a keto acid. Aro ATs are involved in biosynthesis or degradation of AAAs and play important functions in controlling the production of plant hormones and secondary metabolites, such as auxin, tocopherols, flavonoids, and lignin. Most Aro ATs show substrate promiscuity and can accept multiple aromatic and non-aromatic amino and keto acid substrates, which complicates and limits our understanding of their in planta functions. Considering the critical roles Aro ATs play in plant primary and secondary metabolism, it is important to accurately determine substrate specificity and kinetic properties of Aro ATs. This chapter describes various methodologies of protein expression, purification and enzymatic assays, which can be used for biochemical characterization of Aro ATs.

Mass spectrometry imaging-based assays for aminotransferase activity reveal a broad substrate spectrum for a previously uncharacterized enzyme.

Aminotransferases (ATs) catalyze pyridoxal 5'-phosphate-dependent transamination reactions between amino donor and keto acceptor substrates and play central roles in nitrogen metabolism of all organisms. ATs are involved in the biosynthesis and degradation of both proteinogenic and nonproteinogenic amino acids and also carry out a wide variety of functions in photorespiration, detoxification, and secondary metabolism. Despite the importance of ATs, their functionality is poorly understood as only a small fraction of putative ATs, predicted from DNA sequences, are associated with experimental data. Even for characterized ATs, the full spectrum of substrate specificity, among many potential substrates, has not been explored in most cases. This is largely due to the lack of suitable high-throughput assays that can screen for AT activity and specificity at scale. Here we present a new high-throughput platform for screening AT activity using bioconjugate chemistry and mass spectrometry imaging-based analysis. Detection of AT reaction products is achieved by forming an oxime linkage between the ketone groups of transaminated amino donors and a probe molecule that facilitates mass spectrometry-based analysis using nanostructure-initiator mass spectrometry or MALDI-mass spectrometry. As a proof-of-principle, we applied the newly established method and found that a previously uncharacterized Arabidopsis thaliana tryptophan AT-related protein 1 is a highly promiscuous enzyme that can utilize 13 amino acid donors and three keto acid acceptors. These results demonstrate that this oxime-mass spectrometry imaging AT assay enables high-throughput discovery and comprehensive characterization of AT enzymes, leading to an accurate understanding of the nitrogen metabolic network.

A successful case of transarterial chemoembolization for hyperprogressive disease induced by immunotherapy in a patient with unresectable hepatocellular carcinoma.

A fraction of patients with hepatocellular carcinoma (HCC) shows unexpected rapid tumor growth, called hyperprogressive disease (HPD) after the initiation of atezolizumab and bevacizumab (ATZ + BEV). However, little information is available concerning salvage therapy after HPD and the possibility of resuming ATZ + BEV. A 60-year-old woman with unresectable HCCs was treated with transarterial chemoembolization (TACE) and followed by lenvatinib, which showed an unsatisfactory result. Multiple HCCs had been noted in both lobes just before ATZ + BEV treatment. After the initiation of ATZ + BEV, a tumor in the left lobe grew rapidly. The tumor growth kinetics ratio and tumor growth rate ratio of the rapidly growing lesion were 3.76 and 2.02, respectively. Thyroid dysfunction was noted after the initiation of ATZ + BEV. The neutrophil/lymphocyte ratios just before and at 3 weeks after the first ATZ + BEV treatment were 3.89 and 3.5, respectively. Drug-eluting bead (DEB)-TACE using cisplatin was performed for the rapidly growing tumor, which was effective for the targeted HCC in the left lobe as well as multiple HCCs in the right lobe. We were able to resume and continue ATZ + BEV without HPD, which was effective for HCC. We considered that DEB-TACE is an option for treating HPD.

Probiotics suppress nonalcoholic steatohepatitis and carcinogenesis progression in hepatocyte-specific PTEN knockout mice.

Nonalcoholic fatty liver disease (NAFLD), a hepatic characteristic of metabolic syndrome, received significant attention in clinical settings. The multiple-hit theory is one of the proposed mechanisms of NAFLD, and gut dysbiosis is considered a hit. Thus, controlling gut microbiota is a potential target in the management of NAFLD, and probiotics can be used as a treatment agent for NAFLD. The current study aimed to investigate the efficacy of probiotics against nonalcoholic steatohepatitis in a hepatocyte-specific PTEN knockout mouse model that mimics the characteristics of human NAFLD. Probiotics were administered to male knockout mice for 8 or 40 weeks. Next, we assessed hepatic inflammation, fibrosis, carcinogenesis, and oxidative stress. Probiotics were found to reduce serum transaminase levels, NAFLD activity score, and the gene expression of pro-inflammatory cytokines. In addition, they decreased liver fibrosis grade, which was examined via Sirius red staining, gene expression of fibrotic markers, and hydroxyproline. Furthermore, probiotics suppressed the number of liver tumors, particular in HCC. Probiotics reduced oxidative stresses, including glutathione levels, and anti-oxidative stress marker, which may be an underlying mechanism for their beneficial effects. In conclusion, probiotics treatment had beneficial effects against NAFLD and carcinogenesis in hepatocyte-specific PTEN knockout mice.

An automated workflow that generates atom mappings for large-scale metabolic models and its application to Arabidopsis thaliana.

Quantification of reaction fluxes of metabolic networks can help us understand how the integration of different metabolic pathways determines cellular functions. Yet, intracellular fluxes cannot be measured directly but are estimated with metabolic flux analysis (MFA), which relies on the patterns of isotope labeling of metabolites in the network. The application of MFA also requires a stoichiometric model with atom mappings that are currently not available for the majority of large-scale metabolic network models, particularly of plants. While automated approaches such as the Reaction Decoder Toolkit (RDT) can produce atom mappings for individual reactions, tracing the flow of individual atoms of the entire reactions across a metabolic model remains challenging. Here we establish an automated workflow to obtain reliable atom mappings for large-scale metabolic models by refining the outcome of RDT, and apply the workflow to metabolic models of Arabidopsis thaliana. We demonstrate the accuracy of RDT through a comparative analysis with atom mappings from a large database of biochemical reactions, MetaCyc. We further show the utility of our automated workflow by simulating 15 N isotope enrichment and identifying nitrogen (N)-containing metabolites which show enrichment patterns that are informative for flux estimation in future 15 N-MFA studies of A. thaliana. The automated workflow established in this study can be readily expanded to other species for which metabolic models have been established and the resulting atom mappings will facilitate MFA and graph-theoretic structural analyses with large-scale metabolic networks.

Utility of FibroScan-based scoring systems to narrow the risk group of nonalcoholic fatty liver disease with comorbidities.

BACKGROUND: Vibration-controlled transient elastography (VCTE) is proposed as a second step of examination to assess liver fibrosis in patients with nonalcoholic fatty liver disease (NAFLD) after triaging by the fibrosis-4 (FIB-4) index. Recently, VCTE-based scoring systems, including FibroScan-AST (FAST), Agile 3+, and Agile 4, emerged to determine the status of NAFLD. However, the significance of these scoring systems remains unknown in narrowing the high-risk group of NAFLD patients with comorbidities, including hepatocellular carcinoma (HCC) and esophagogastric varices (EGV). AIM: To clarify the significance of VCTE-based scoring systems to narrow the high-risk group of NAFLD patients with comorbidities. METHODS: We performed a cross-sectional study to investigate the usefulness of VCTE-based scoring systems and other fibrosis markers to narrow the high-risk group of patients with NAFLD. FIB-4 index was used for the first triage. Risk groups of FAST, Agile 3+, and Agile 4 were stratified according to the published data. Among the 191 patients with NAFLD, there were 26 (14%) and 25 patients (13%) with HCC and EGV, respectively. RESULTS: When 1.3 was used as a cutoff value, the FIB-4 index narrowed the risk group to 120 patients, in which all patients with HCC and/or EGV were included. High risk group of Agile 3+ could subsequently narrow the risk group. The prevalence of HCC and EGV at this step were 33% (26/80) and 31% (25/80), respectively. In further narrowing of EGV, Agile 4 aggregated the patients with EGV into 43 patients, of whom 23 (53%) had EGV. FAST failed to narrow the risk group of patients with comorbidities. When 2.6 was used as a cutoff value of the FIB-4 index, three patients with HCC and two patients with EGV were missed at the first triage. CONCLUSION: Agile 3+ and Agile 4 are useful to narrow the NAFLD patient group, in which patients may have HCC and/or EGV.

Point mutations that boost aromatic amino acid production and CO2 assimilation in plants.

Aromatic compounds having unusual stability provide high-value chemicals and considerable promise for carbon storage. Terrestrial plants can convert atmospheric CO2 into diverse and abundant aromatic compounds. However, it is unclear how plants control the shikimate pathway that connects the photosynthetic carbon fixation with the biosynthesis of aromatic amino acids, the major precursors of plant aromatic natural products. This study identified suppressor of tyra2 (sota) mutations that deregulate the first step in the plant shikimate pathway by alleviating multiple effector-mediated feedback regulation in Arabidopsis thaliana. The sota mutant plants showed hyperaccumulation of aromatic amino acids accompanied by up to a 30% increase in net CO2 assimilation. The identified mutations can be used to enhance plant-based, sustainable conversion of atmospheric CO2 to high-energy and high-value aromatic compounds.

Evolutionary origin and functional diversification of aminotransferases.

Aminotransferases (ATs) are pyridoxal 5'-phosphate-dependent enzymes that catalyze the transamination reactions between amino acid donor and keto acid acceptor substrates. Modern AT enzymes constitute ∼2% of all classified enzymatic activities, play central roles in nitrogen metabolism, and generate multitude of primary and secondary metabolites. ATs likely diverged into four distinct AT classes before the appearance of the last universal common ancestor and further expanded to a large and diverse enzyme family. Although the AT family underwent an extensive functional specialization, many AT enzymes retained considerable substrate promiscuity and multifunctionality because of their inherent mechanistic, structural, and functional constraints. This review summarizes the evolutionary history, diverse metabolic roles, reaction mechanisms, and structure-function relationships of the AT family enzymes, with a special emphasis on their substrate promiscuity and multifunctionality. Comprehensive characterization of AT substrate specificity is still needed to reveal their true metabolic functions in interconnecting various branches of the nitrogen metabolic network in different organisms.

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase as the gatekeeper of plant aromatic natural product biosynthesis.

The shikimate pathway connects the central carbon metabolism with the biosynthesis of aromatic amino acids-l-tyrosine, l-phenylalanine, and l-tryptophan-which play indispensable roles as precursors of numerous aromatic phytochemicals. Despite the importance of the shikimate pathway-derived products for both plant physiology and human society, the regulatory mechanism of the shikimate pathway remains elusive. This review summarizes the recent progress and current understanding on the plant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHP synthase or DHS) enzymes that catalyze the committed reaction of the shikimate pathway. We particularly focus on how the DHS activity is regulated in plants in comparison to those of microbes and discuss potential roles of DHS as the critical gatekeeper for the production of plant aromatic compounds.